Breeding Novel Chemistry in Willow: New Hetero Diels–Alder Cyclodimers from Arbusculoidin and Salicortin Suggest Parallel Biosynthetic Pathways

Investigation of phenolic glycosides extracted from Salix germplasm revealed that arbusculoidin (benzyl 1-O-β-D-glucopyranosyl-1-hydroxy-6-oxo-2-cyclohexenyl carboxylate) and its enolic 6-glycoside isomer, isoarbusculoidin are widespread across the Salix family. Analysis of natural hybrid species and progeny from a willow breeding programme demonstrated that the putative biosynthetic pathway leading to the salicinoid family of phenolic glycosides runs in parallel to a “benzyl”- based pathway to arbusculoidin. Introduction of a known Diels-Alder reaction trait from Salix miyabeana, as well as an acylation trait, into progeny containing both salicyl- and benzyl- pathways caused the formation of all possible hetero-cyclodimers, from mixtures of reactive dienone (acyl)glycosides that participate in cross-over reactions. As well as providing access to new analogues of the anti-cancer dimer, miyabeacin, analysis of breeding progeny also indicates that these dienone (acyl)glycosides dienones are stable in planta. Although the immediate biosynthetic precursors of these compounds remain to be defined, the results suggest the (acyl)glycosylation reactions may occur later in the pathway than previously suggested by in vitro work on cloned UGT enzymes.